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The construction of highways is responsible for access to previously protected areas, resulting in changes in landscape and dynamics of the animal populations that live in these areas. These enterprises are the major responsible for the mortality of wild animals, surpassing hunting and even the trafficking of animals. The objective of this study was to make a list that reflects the diversity of amphibians and reptile’s road-killed along the BR-040, a highway that crosses the threaten lowland Atlantic Forest in Southeastern region of Brazil, including the use of microhabitats, lifestyle, activity pattern, reproductive cycles, and possible rare or endangered species. The study area consists of 180,4 km of highways. Monitoring began in 2006 and continues to the present day. A total of 1,410 individuals from 60 species were recorded in this study. The reptiles were more frequent in number of individuals and species. The commonest species recorded were Crotalus durissus and Dipsas mikanii. We have registered a single endangered species: Ranacephala hogei. The highest rates of road-kill were recorded during the wet season. Road-kills of fauna is a major threat to species, studies are of great importance to define plans that seek to mitigate the effects generated by these enterprises. [ABSTRACT FROM AUTHOR]